Japanese encephalitis virus (JEV), a mosquito-borne virus, causes severe clinical symptoms in humans in the Asian-Pacific region, where it circulates in a primary transmission cycle among Culex tritaeniorhynchus mosquitoes, domestic swine (Sus scrofa domesticus), and wading birds. We report here an anomalous result that mosquito-borne JEV was detected in unfed host-questing ticks collected from the field in Japan. JEV genomic RNA was detected in four pools of Haemaphysalis flava nymphs collected in November and December 2019, and March 2020, when Cx. tritaeniorhynchus adults were not presumed to be active. Moreover, JEV antigenomic RNA was detected in some JEV-positive tick samples, suggesting virus replication in ticks. However, taken together with no infectious virus isolated, the possibility that the antigenomic RNA was derived from the undigested bloodmeal source in ticks cannot be ruled out. Thus, the role of the ticks as a natural reservoir for JEV remains to be confirmed.